Inhibitors of Tyrosine Kinase-Dependent Signaling as Anti-Cancer Agents

酪氨酸激酶依赖性信号传导抑制剂作为抗癌药物

• 批准号: 10702292
• 负责人: TERRENCE BURKE
• 金额: $ 45.43万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702292
• 关键词: Advanced Development; Affinity; Aldehydes; Amines; Amino Acid Sequence; Antineoplastic Agents; Area; Benzoic Acids; Binding; Biochemical; C-terminal; Catalytic Domain; Cell division; Clinical Trials; Collaborations; Complex; Crystallization; DNA; DNA Repair Enzymes; Development; Docking; Drug resistance; Elements; Exhibits; Goals; Hot Spot; Hydrolysis; Imidazole; Label; Laboratories; Legal patent; Libraries; Ligand Binding; Ligand Binding Domain; Ligands; Link; Malignant Neoplasms; Mediating; Molecular; Molecular Target; N-terminal; Nitrogen; Oximes; PLK1 gene; Parents; Peptides; Pharmaceutical Preparations; Pharmacology; Phosphopeptides; Phosphoserine; Phosphothreonine; Phosphotransferases; Phthalic Acids; Physiological; Play; Polo-Box Domain; Process; Prognosis; Proteins; Reaction; Reagent; Reporting; Roentgen Rays; Role; Serine; Signal Transduction; Skeleton; Structure; Substrate Interaction; TOP1 gene; Technology; Threonine; Time; Type I DNA Topoisomerases; Tyrosine; Tyrosine Kinase Inhibitor; Up-Regulation; Variant; Work; analog; antagonist; anti-cancer; anti-cancer therapeutic; base; cancer therapy; chemical stability; cytotoxicity; design; improved; inhibitor; inorganic phosphate; nanomolar; overexpression; peptide structure; phosphodiester; polo-like kinase kinase 1; protein kinase inhibitor; protein protein interaction; repaired; small molecule; therapeutic development; therapy development; tool; tyrosyl-DNA phosphodiesterase

defined as a molecular target for anti-cancer therapy development. The Plk1 plays a central role in cell division and upregulation of Plk1 activity appears to be closely associated with aggressiveness and poor prognosis of several cancers. This protein is overexpressed in many cancers and its inhibition can result in antiproliferative effects. Plk1 requires the coordinated actions of both an N-terminal kinase domain (KD), which executes its catalytic function and a C-terminal polo-box domain (PBD), which engages in protein - protein interactions (PPIs) with phosphoserine (pS) and phosphothreonine (pT)-containing sequences. Although Plk1 KD-directed agents are currently in clinical trials for the treatment of cancers, issues related to cytotoxicity have arisen that may result from off-target effects. Targeting protein - protein interactions (PPIs) has emerged as an important area for anticancer therapeutic development. In the case of phospho-dependent PPIs, such as the Plk1 PBD, a phosphorylated protein residue can provide high-affinity recognition and bind to target protein hot spots. Starting from the 5-mer phosphopeptide "PLHSpT" and in collaboration with the NCI laboratory of Dr. Kyung Lee and the MIT laboratory of Dr. Michael Yaffe, we initially identified inhibitory peptides that showed from 1000- to more than 10,000-fold improved PBD-binding affinity. X-ray co-crystal structures of these peptides bound to Plk1 PBD indicated unanticipated modes of binding, which take advantage of a "cryptic" binding channel that is not present in the non-liganded PBD or engaged by the parent pentamer phosphopeptide. The cryptic pocket is accessed by means of a phenylalkyl moiety attached to the N(pi) nitrogen of the His imidazole ring. Multivalency can be a powerful means to achieve highly potent and selective ligand-protein interactions. The selectivity and affinity of protein kinase (PK) inhibitors can be greatly increased by linking an element that binds within the ATP-binding cleft together with a component that binds exterior to the cleft. When the secondary component accesses ancillary regulatory domains, the resulting ligand may be described as being intramolecular "bivalent." We have undertaken work to develop bivalent ligands, designed to simultaneously engage both KD and PBD regions of Plk1. This has resulted in bivalent constructs exhibiting more than 100-fold Plk1 affinity enhancement relative monovalent PBD-binding ligands, which had until this time, exhibited among the highest PBD-binding affinities yet reported. Startlingly, and in contradiction to widely accepted notions of KD-PBD interactions, we have found that extremely high affinities can be retained even with minimal linkers between KD and PBD-binding components. In addition to significantly advancing the development of PBD-binding ligands, our findings may cause a rethinking of the structure-function of Plk1 and potential implications for the physiological roles played by this kinase. Objective Two: Tyrosyl-DNA phosphodiesterase 1 (TDP1) it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of TOP1 and the -phosphate of its DNA substrate. Blocking TDP1 function would be an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. TDP1 inhibitors would represent a new and potentially promising class of anticancer agents that could be used with TOP1 inhibitors in anticancer therapy. Although there have been reports of TDP1 inhibitors, there is a pressing need for the discovery of effective and specific TDP1 inhibitors for which there is validated binding and a defined mechanism of actions. In collaboration with the NCI laboratories of Dr. David Waugh and Dr. Yves Pommier, used an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. More recently, in collaboration with the NCI laboratory of Dr. Jay Schneekloth, we performed a TDP1 small molecule microarray screen of over 21,000 drug-like molecules in a small molecules microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified 109 hits from 21,000 compounds (0.5% hit rate) and arrived at a preferred TDP1-binding motif. Among the hits were structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines, which we demonstrated functioned as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke-Blackburn-Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. We are currently elaborating the structure of the parent SMM-derived platform by adding functionality that extends into the peptide and DNA substrate binding regions. We are using a "click"-based oxime diversification strategy that we have used successfully in several applications to optimize the binding interactions of parent ligands. A key to this approach is its ability to take a single synthetic parent construct and easily diversity it using a library of readily obtainable aldehyde reagents. In this work, we are modifying our SMM-derived platforms by adding aminooxy handles. This yielded two parent aminooxy-containing constructs. The benzoic acid moieties of these constructs are intended to bind within the catalytic site phosphoryl-binding pocket while the aminooxy groups are situated so that the resulting oxime derivatives would access the DNA or peptide substrate-binding channels. In this way, we were able to rapidly interrogate the structures of approximately 500 oxime derivatives. The most promising compounds (low micromolar IC50 values) were further derivatized to increase the chemical stability of the parent oxime linkages. Through this process, we have been able to achieve TDP1 inhibitors with nanomolar potencies. We have recently received the crystal structure of oxime-derived inhibitors bound to the TDP1 catalytic site and it appears that they bind in a fashion that is similar to what was predicted by our molecular docking studies. Going forward our goal is to derive validated inhibitors with defined binding interactions. These inhibitors will provide pharmacological tools for studying the biochemical effects of competitively inhibiting TDP1 function in cellular settings. Our work should advance the general field of TDP1 inhibitor development. A PCT patent application has recently been filed covering aspects of this technology.

被定义为抗癌治疗开发的分子靶点。 Plk1 在细胞分裂中发挥核心作用，Plk1 活性的上调似乎与多种癌症的侵袭性和不良预后密切相关。这种蛋白质在许多癌症中过度表达，抑制它可以产生抗增殖作用。 Plk1 需要 N 端激酶结构域 (KD) 和 C 端 polo-box 结构域 (PBD) 的协调作用，其中 N 端激酶结构域执行其催化功能，C 端 polo 盒结构域 (PBD) 参与与磷酸丝氨酸 (pS) 的蛋白质-蛋白质相互作用 (PPI)和含磷酸苏氨酸（pT）的序列。 尽管 Plk1 KD 导向药物目前正处于治疗癌症的临床试验中，但可能因脱靶效应而出现与细胞毒性相关的问题。 靶向蛋白质-蛋白质相互作用（PPI）已成为抗癌治疗开发的一个重要领域。对于磷酸依赖性 PPI，例如 Plk1 PBD，磷酸化的蛋白质残基可以提供高亲和力识别并与目标蛋白质热点结合。从 5 聚体磷酸肽“PLHSpT”开始，与 Kyung Lee 博士的 NCI 实验室和 Michael Yaffe 博士的 MIT 实验室合作，我们初步鉴定了抑制性肽，其 PBD 改善了 1000 至 10,000 倍以上-结合亲和力。这些肽与 Plk1 PBD 结合的 X 射线共晶结构表明了意想不到的结合模式，该模式利用了非配体 PBD 中不存在的或与亲本五聚体磷酸肽接合的“神秘”结合通道。 通过连接至组氨酸咪唑环的 N(pi) 氮的苯基烷基部分可进入隐秘袋。多价可以是实现高效和选择性配体-蛋白质相互作用的有力手段。通过将结合在 ATP 结合裂口内的元件与结合在裂口外部的成分连接在一起，可以大大提高蛋白激酶 (PK) 抑制剂的选择性和亲和力。当次要成分进入辅助调节结构域时，所得配体可被描述为分子内“二价”。我们已经着手开发二价配体，旨在同时接合 Plk1 的 KD 和 PBD 区域。这导致二价构建体相对单价 PBD 结合配体表现出超过 100 倍的 Plk1 亲和力增强，迄今为止，二价构建体表现出迄今为止报道的最高的 PBD 结合亲和力。令人惊讶的是，与广泛接受的 KD-PBD 相互作用概念相矛盾的是，我们发现即使 KD 和 PBD 结合成分之间的连接体最少，也可以保留极高的亲和力。除了显着推进 PBD 结合配体的发展之外，我们的发现可能会引起人们对 Plk1 结构功能的重新思考以及对该激酶所发挥的生理作用的潜在影响。目标二：酪氨酰-DNA磷酸二酯酶1（TDP1），它能够通过修复TOP1与DNA失速的共价复合物来降低I型拓扑异构酶（TOP1）抑制剂的抗癌作用。它通过促进 TOP1 的 Y723 残基与其 DNA 底物的磷酸之间的磷酸二酯键的水解来实现这一点。 阻断 TDP1 功能将是增强 TOP1 抑制剂疗效和克服耐药性的一种有吸引力的方法。 TDP1 抑制剂将代表一类新的、有潜力的抗癌药物，可与 TOP1 抑制剂一起用于抗癌治疗。尽管已有 TDP1 抑制剂的报道，但迫切需要发现有效且特异性的 TDP1 抑制剂，并对其进行验证的结合和明确的作用机制。与 NCI 实验室的 David Waugh 博士和 Yves Pommier 博士合作，使用包含 600 多个片段的 X 射线晶体屏幕来识别 TDP1 催化口袋内结合的邻苯二甲酸和羟基喹啉基序的小分子变异。然而，这些化合物中的大多数表现出有限的（毫摩尔）TDP1 抑制效力。最近，我们与 Jay Schneekloth 博士的 NCI 实验室合作，对小分子微阵列 (SMM) 格式的 21,000 多种药物样分子进行了 TDP1 小分子微阵列筛选，以确定它们结合 Alexa Fluor 647 (AF647) 的能力-标记为TDP1。筛选从 21,000 种化合物中鉴定出 109 个命中（命中率 0.5%），并得出了首选的 TDP1 结合基序。其中包括结构相似的 N,2-二苯基咪唑并[1,2-a]吡嗪-3-胺，我们证明其具有 TDP1 结合剂和催化抑制剂的功能。然后，我们使用一锅 Groebke-Blackburn-Bienayme 多组分反应探索了核心杂环骨架，并得到了具有更高抑制效力的类似物。解析一部分化合物的 TDP1 共晶结构显示它们在 TDP1 催化位点上结合，同时模拟底物相互作用。我们目前正在通过添加延伸至肽和 DNA 底物结合区域的功能来详细阐述母体 SMM 衍生平台的结构。我们正在使用基于“点击”的肟多样化策略，我们已在多个应用中成功使用该策略来优化亲本配体的结合相互作用。这种方法的关键是它能够采用单一的合成亲本构建体，并使用易于获得的醛试剂库轻松地对其进行多样性。在这项工作中，我们通过添加氨氧基手柄来修改我们的 SMM 衍生平台。这产生了两个亲本含氨氧基构建体。这些构建体的苯甲酸部分旨在结合在催化位点磷酰基结合袋内，而氨氧基的位置使得所得肟衍生物能够进入DNA或肽底物结合通道。通过这种方式，我们能够快速解析大约 500 种肟衍生物的结构。最有前途的化合物（低微摩尔 IC50 值）被进一步衍生化，以提高母体肟键的化学稳定性。通过这个过程，我们已经能够获得具有纳摩尔效力的 TDP1 抑制剂。我们最近收到了与 TDP1 催化位点结合的肟衍生抑制剂的晶体结构，看来它们的结合方式与我们的分子对接研究预测的相似。展望未来，我们的目标是衍生出具有明确的结合相互作用的经过验证的抑制剂。这些抑制剂将为研究细胞环境中竞争性抑制 TDP1 功能的生化效应提供药理学工具。我们的工作应该推进 TDP1 抑制剂开发的一般领域。最近提交的 PCT 专利申请涵盖了该技术的各个方面。